臺灣博碩士論文加值系統

中文摘要
腺嘌呤核二磷酸核糖化因子是真核細胞中小鳥糞嘌呤結合蛋白超級家族中的一員，廣泛地存在於從酵母菌至人類的所有真核生物細胞中。腺嘌呤核二磷酸核糖化因子蛋白具有輔助霍亂毒素所引起的異三體鳥糞嘌呤結合蛋白阿爾發次單元體的腺嘌呤核二磷酸核糖化作用。目前已知腺嘌呤核二磷酸核糖化因子蛋白主要參與調控細胞內由膜所構成的胞器系統之間的囊泡生成及傳遞，活化丁磷脂質水解…等方面。在本論文中，我們嘗試去探討酵母菌的第三腺嘌呤核二磷酸核糖化因子在細胞內的生理功能以及在細胞內的分佈位置。酵母菌的第一腺嘌呤核二磷酸核糖化因子與第二腺嘌呤核二磷酸核糖化因子是酵母菌的必須基因;然而酵母菌第三腺嘌呤核二磷酸核糖化因子卻與酵母菌的生存無關，也不負責由內質網到高基氏體的囊泡傳遞、一般的持續內噬作用，或由受體所調控的內噬作用。由次細胞內的離心分離法與免疫螢光染色分析法發現，酵母菌第三腺嘌呤核二磷酸核糖化因子專一地分佈在液泡膜上。由過量表現的蛋白分析，酵母菌第三腺嘌呤核二磷酸核糖化因子的分佈模式和與其最相似的人類第六腺嘌呤核二磷酸核糖化因子過量表現在正常大鼠的腎臟細胞與海拉細胞間的分佈模式不相像。這種獨特的分佈模式可能表示酵母菌第三腺嘌呤核二磷酸核糖化因子在細胞內扮演了與眾不同的角色。在代表酵母菌第三腺嘌呤核二磷酸核糖化因子與鳥糞嘌呤核三磷酸結合態的醯安穀安酸轉換成白安酸的變異蛋白，以及代表酵母菌的第三腺嘌呤核二磷酸核糖化因子與鳥糞嘌呤核二磷酸結合態的酥安酸轉換成醯安天門冬酸的變異蛋白的分析上，我們發現不同狀態的酵母菌第三腺嘌呤核二磷酸核糖化因子分佈在細胞中的不同位置。同時也發現代表酵母菌第三腺嘌呤核二磷酸核糖化因子無法被肉荳蔻酸化的甘安酸轉換成丙安酸的突變異蛋白仍然可以與特定的膜類構造結合在一起，這和先前推測腺嘌呤核二磷酸核糖化因子必須經由肉荳蔻酸化才能夠結合到膜膜類構造上的假設不同。在免疫螢光染色中，該種突變蛋白則是分佈在近細胞膜的小顆粒囊泡上。在酵母菌第一腺嘌呤核二磷酸核糖化因子與酵母菌第三腺嘌呤核二磷酸核糖化因子的嵌合體蛋白研究中，我們發現酵母菌第三腺嘌呤核二磷酸核糖化因子可能本身既具有與特定的膜類構造結合的能力。

Abstract
The ADP-ribosylation factor (ARF) GTP-binding proteins are ubiquitous, eukaryotic regulators of virtually every step of vesicular membrane traffic. Based on size, phylogenetic analysis, amino acid sequence, and gene struc-ture, mammalian ARFs fall into three classes (class I, ARFs 1, 2, 3; class II, ARFs 4, 5; class III, ARF6). Two ARF genes (yARF1, yARF2) are essential in yeast and the lethality resulting from either overexpression or underexpression (deletion) of Arf genes has previously been ascribed to dysregulation of the secretory process. A third yeast ARF (yARF3) is more identical (60%) to that the class III mammalian ARF6. yARF3 is not essential for cell viability and is not required for ER to Golgi protein transport, fluid-phase endocytosis, or re-ceptor mediated endocytosis. By subcellular fractionation and immunofluo-rescence, we found that yARF3 is specifically associated with and localized to the vacuole membrane. This pattern of localization of the endogenous pro-tein was unlike to the localization of ARF6 when overexpressed in NRK, or HeLa, cells. Localization of yARF3 to the vacuole membrane suggests a unique cellular role for this isoform at the plasma membrane although failure to find endogenous yARF3 on endocytic structures. yARF3 mutants Q71L and T31N, which are predicted to be in either the GTP- or GDP-bound state, were expressed and characterized, respectively. Our results indicated both forms of mutants were also localized to plasma membrane or tubulovesicular com-partment near vacuole. These results suggest that although all the ARF proteins play fundamental and critical roles in cellular function, they are in-volved in different vesicular transport processes. We also demonstrated that the G2A yARF3 mutant, which was a non-myristoylated mutant, localized on small endosomes nearby the cytoplasmic membrane. Our results provide evidence that the N'-terminal myristoylation is not the only one critical require-ment for yARF3 to associate with specific membrane. Furthermore, accord-ing to the distribution of chimeras from yARF1 and yARF3, chimera which has the N'-terminus of yARF3 was found on plasma membrane whereas the counterpart which has the N'-terminus of yARF1 was punctuated in cytoplasm.

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Reference
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